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www.biopharminternational.com July 2021 BioPharm International eBook 23 where development timelines are forc- ing the use of generic reagents, those reagents should be checked for speci- ficity and actual HCP coverage and the results be conf irmed by an unbiased approach such as mass spectrometry. Quantification of HCPs by LC-MS is an orthogonal and unbiased approach to detection by ELISA. Importantly, because well-characterized protein and peptide standards can be spiked into test articles at defined concentrations, LC-MS provides an accurate, reliable quantitative readout of actual HCP con- tent. Furthermore, LC-MS provides for identification of the individual proteins detected, potentially guiding the refine- ment of AAV purification strategies on a faster timescale than is possible by prod- uct-specific ELISA development. A f ter hav ing t he immu noassay detection gaps identified by MS, those gaps can be closed by an approach that uses a combination of in-silico immunogenicity prediction, peptide synthesis, and a fast-track immuni- zation program. The goal is to gener- ate highly specific antibodies against the individual HCP deficiencies of an existing immunoassay. Biopharmaceutical Analysis Impurity Analysis Figure 3. Coverage evaluation of kit reagents. Linear coverage by 2D sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blot. HCP is host cell proteins. HEK 293 cell lysate Total Protein Silver Stain Western Blot using commercially available anti-HEK293 HCP antibodies (goat) Coverage 28% Figure 4. Fast-track closure of detection gaps by peptide immunization. KHL is keyhole limpet hemocyanin. Synthesis of predicted peptides Coupling of peptides to KLH Keyhole Limpet: Megathura crenulata KLH Immunization of peptide-carrier complex Analysis of protein sequence for prediction of immunogenic peptides KL H KL H KL H Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y YY Y Y Y Y Y Y Y Y Y Y Y Y YY Y Y Y Y Y Y Pooling of antibodies from different immunizations